The present invention relates in general to herbicide resistance in plants, and more particularly to a new class of phosphonate metabolizing genes and methods of using these genes for improving plant tolerance to phosphonate herbicides.
Phosphorous containing organic molecules can be naturally occurring or synthetically derived. Organic molecules containing phosphorous-carbon (Cxe2x80x94P) bonds are also found naturally or as synthetic compounds, and are often not rapidly degraded, if at all, by natural enzymatic pathways. Synthetic organophosphonates and phosphinates, compounds that contain a direct carbon-phosphorous (Cxe2x80x94P) bond in place of the better known carbon-oxygen-phosphorous linkage of phosphate esters (Metcalf et al., Gene 129:27-32, 1993), have thus been widely used as insecticides, antibiotics, and as herbicides (Chen et al., J. Biol. Chem. 265:4461-4471, 1990; Hilderbrand et al., The role of phosphonates in living systems, Hilderbrand, R. L., ed, pp. 5-29, CRC Press, Inc., Boca Raton, Fla., 1983). Phosphonates are ubiquitous in nature, and are found alone and in a diversity of macromolecular structures in a variety of organisms (Jiang et al., J. Bacteriol. 177:6411-6421, 1995). Degradation of phosphonate molecules proceeds through a number of known routes, a Cxe2x80x94P lyase pathway, a phosphonatase pathway, and a Cxe2x80x94N hydrolysis pathway (Wanner, Biodegradation 5:175-184, 1994; Barry et al., U.S. Pat. No. 5,463,175, 1995). Bacterial isolates capable of carrying out these steps have been characterized (Shinabarger et al., J. Bacteriol. 168:702-707, 1986; Kishore et al., J. Biol. Chem. 262:12,164-12,168, 1987; Pipke et al., Appl. Environ. Microbiol. 54:1293-1296,1987; Jacob et al., Appl. Environ. Microbiol. 54:2953-2958, 1988; Lee et al., J. Bacteriol. 174:2501-2510, 1992; Dumora et al., Biochim. Biophys. Acta 997:193-198, 1989; Lacoste et al., J. Gen. Microbiol. 138:1283-1287, 1992). However, with the exception of phosphonatase and glyphosate oxidase (GOX), other enzymes capable of carrying out these reactions have not been characterized.
Several studies have focused on the identification of genes required for Cxe2x80x94P lyase degradation of phosphonates. Wackett et al. (J. Bacteriol. 169:710-717, 1987) disclosed broad substrate specificity toward phosphonate degradation by Agrobacterium radiobacter and specific utilization of glyphosate as a sole phosphate source. Shinabarger et al. and Kishore et al. disclosed Cxe2x80x94P lyase degradation of the phosphonate herbicide, glyphosate, to glycine and inorganic phosphate through a sarcosine intermediate by Pseudomonas species.
E. coli B strains had previously been shown to be capable of phosphonate utilization (Chen et al.), whereas E. coli K-12 strains were incapable of phosphonate degradation. However, K-12 strains were subsequently shown to contain a complete, though cryptic, set of genes (psiD or phn) capable of phosphonate utilization (Makino et al.), as mutants were easily selected by growth on low phosphate media containing methyl- or ethyl-phosphonate as sole phosphorous sources. Such K-12 strains adapted for growth on methyl- or ethylphosphonate were subsequently shown to be able to utilize other phosphonates as sole phosphorous sources (Wackett et al., J. Bacteriol. 169:1753-1756, 1987).
Avila et al. (J. Am. Chem. Soc. 109:6758-6764, 1987) were interested in the mechanistic appraisal of biodegradative and detoxifying processes as related to aminomethyl-phosphonates, including elucidating the intermediates, products, and mechanisms of the degradative dephosphorylation process. Avila et al. studied the formation of dephosphorylated biodegradation products from a variety of aminophosphonate substrates in E. coli K-12 cultures previously adapted to growth on ethylphosphonate. Furthermore, Avila et al. utilized N-acetyl-AMPA (N-acetyl-amino-methyl-phosphonate) as a sole phosphate source in some of their studies in order to show that acetylated AMPA was not inhibitory to Cxe2x80x94P bond cleavage. In addition, Avila et al. noted that N-acetyl-AMPA was able to serve as a sole phosphate source during E. coli K-12 growth, however, they did not observe N-acetyl-AMPA formation when AMPA was used as a sole phosphate source. Their results indicated that AMPA was not a substrate for acetylation in E. coli. 
Chen et al. identified a functional psiD locus from E. coli B by complementation cloning into an E. coli K-12 strain deficient for phosphonate utilization, which enabled the K-12 strain to utilize phosphonate as a sole phosphate source (J. Biol. Chem. 265:4461-4471, 1990). Chen et al. thus disclosed the DNA sequence of the psiD complementing locus, identified on a 15.5 kb BamHI fragment containing 17 open reading frames designated phnA-phnQ, comprising the E. coli B phn operon. The cryptic phn (psiD) operon from E. coli K-12 was subsequently found to contain an 8-base pair insertion in phnE. The resulting frameshift in phnE not only results in defective phnE gene product, but also apparently causes polar effects on the expression of downstream genes within the operon, which prevent phosphonate utilization (Makino et al., J. Bacteriol. 173:2665-2672, 1991). The operon has been more accurately described to contain the genes phnC-phnP by the work of Makino et al. Further research has been directed to understanding the nature of the function of each of the genes within this operon (Chen et al., J. Biol. Chem. 265:4461-4471, 1990; Makino et al., J. Bacteriol. 173:2665-2672, 1991; Wanner et al., FEMS Microbiol. Lett. 100:133-140, 1992; Metcalf et al., Gene 129:27-32, 1993; Ohtaki et al., Actinomyceteol. 8:66-68, 1994). In all of these efforts, the phnO gene has been implicated as a regulatory protein based on its similarity to other nucleotide binding proteins containing structural helix-turn-helix motifs. Furthermore, mutagenesis of genes in the phn operon demonstrated that phnO was not required for phosphonate utilization, further supporting the proposed regulatory function for this gene (Metcalf et al., J. Bacteriol. 173:587-600, 1991), at least for the phosphonates tested. Homologous phn sequences have been identified from other bacteria, including a gene substantially similar to E. coli phnO, isolated from S. griseus, using nucleotide sequences deduced from those in the E. coli phnO gene (Jiang et al., J. Bacteriol. 177:6411-6421, (1995); McGrath et al., Eur. J. Biochem. 234:225-230, (1995); Ohtaki et al., Actinomyceteol. 8:66-68, (1994)). However, no function other than as a regulatory factor has been proposed for phnO. A regulatory role for phnO in the CP lyase operon has been cited again in a recent review (Berlyn, Microbiol. Molec. Biol. Rev. 62:814-984, 1998).
Advances in molecular biology, and in particular in plant sciences in combination with recombinant DNA technology, have enabled the construction of recombinant plants which contain nonnative genes of agronomic importance. Furthermore, when incorporated into and expressed in a plant, such genes desirably confer some beneficial trait or characteristic to the recombinant plant. One such trait is herbicide resistance. A recombinant plant capable of growth in the presence of a herbicide has a tremendous advantage over herbicide-susceptible species. In addition, herbicide tolerant plants provide a more cost effective means for agronomic production by reducing the need for tillage to control weeds and volunteers.
Chemical herbicides have been used for decades to inhibit plant metabolism, particularly for agronomic purposes as a means for controlling weeds or volunteer plants in fields of crop plants. A class of herbicides which have proven to be particularly effective for these purposes are known as phosphonates or phosphonic acid herbicides. Perhaps the most agronomically successful phosphonate herbicide is glyphosate (N-phosphono-methyl-glycine).
Recombinant plants have been constructed which are tolerant to the phosphonate herbicide glyphosate. When applied to plants, glyphosate is absorbed into the plant tissues and inhibits aromatic amino acid formation, mediated by an inhibition of the activity of the plastid-localized 5-enolpyruvyl-3-phosphoshikimic acid synthase enzyme, also known as EPSP synthase or EPSPS, an enzyme generally thought to be unique to plants, bacteria and fungi. Recombinant plants have been transformed with a bacterial EPSPS enzyme which is much less sensitive to glyphosate inhibition. Therefore, plants expressing this bacterial EPSPS are less sensitive to glyphosate, and are often characterized as being glyphosate tolerant. Therefore, greater amounts of glyphosate can be applied to such recombinant plants, ensuring the demise of plants which are susceptible or sensitive to the herbicide. However, other genes have been identified which, when transformed into a plant genome, encoding enzymes which also provide glyphosate tolerance. One such enzyme has been described as GOX, or glyphosate-oxidoredutase. GOX functions in providing protection to plants from the phosphonate herbicide glyphosate by catalyzing the degradation of glyphosate to aminomethyl phosphonic acid (AMPA) and glyoxylate. AMPA produced as a result of glyphosate degradation can cause bleaching and stunted or depressed plant growth, among other undesireable characteristics. Many plant species are also sensitive to exogeneously applied AMPA, as well as to endogenous AMPA produced as a result of GOX mediated glyphosate herbicide degradation. No method has been described which discloses the protection of plants from applications of phosphonate herbicides such as AMPA.
Barry et al. (U.S. Pat. No. 5,633,435) disclose genes encoding EPSP synthase enzymes which are useful in producing transformed bacteria and plants which are tolerant to glyphosate as a herbicide, as well as the use of such genes as a method for selectively controlling weeds in a planted transgenic crop field. Barry et al. (U.S. Pat. No. 5,463,175) disclose genes encoding glyphosate oxidoreductase (GOX) enzymes useful in producing transformed bacteria and plants which degrade glyphosate herbicide as well as crop plants which are tolerant to glyphosate as a herbicide. Barry et al. (U.S. Pat. No. 5,463,175) disclosed the formation of AMPA as a product of GOX mediated glyphosate metabolism.AMPA has been reported to be much less phytotoxic than glyphosate for most plant species (Franz, 1985) but not for all plant species (Maier, 1983; Tanaka et al., 1986). Co-expression of a gene encoding a protein capable of neutralizing or metabolizing AMPA produced by glyphosate degradation would provide a substantial improvement over the use of GOX alone. Thus, a method for overcoming sensitivity to AMPA formation as a result of glyphosate degradation, or a method for resistance to AMPA when used as a herbicide or as a selective agent in plant transformation methods, would be useful for providing enhanced or improved herbicide tolerance in transgenic plants and in other organisms sensitive to such compounds.
The use of glyphosate as a chemical gametocide has been described (U.S. Pat. No. 4,735,649). Therein, it is disclosed that glyphosate can, under optimal conditions, kill about 95% of male gametes, while leaving about 40-60% of the female gametes capable of fertilization. In addition, a stunting effect was typically observed at the application levels disclosed, shown by a reduction in the size of the plant and by a minor amount of chlorosis. Thus, a major drawback of using glyphosate as a gametocide, as is generally true with most gametocides, is the phytotoxic side effects resulting from lack of sufficient selectivity for male gametes. These phytotoxic manifestations may be effectuated by AMPA production in transgenic plants expressing GOX after treatment with glyphosate. Therefore, it would be advantageous to provide a method for preventing the stunting effect and chlorosis as side effects of using glyphosate as a gametocide in transgenic plants expressing GOX. Furthermore, a more effective method would optimally kill more than 95% of male gametes or prevent male gametes from maturing and would leave greater than 60% of female gametes substantially unaffected. It is believed that tissue specific co-expression of GOX with a transacylase gene encoding an enzyme capable of N-acylation of AMPA would achieve this goal.
It has now been discovered that the E. coli phnO gene encodes an enzyme having transacylase, acyltransferase, or Acyl-CoA transacylase activity in which a preferred substrate is a phosphonate displaying a terminal amine, and in particular amino-methyl-phosphonic acid (AMPA). The transfer of an acyl group from an Acyl-CoA to the free terminal amine of AMPA results in the formation of an N-acylated AMPA. Plants are not known to acylate AMPA to any great extent, and some plants have been shown to be sensitive to AMPA and insensitive to acyl-AMPA. Thus, expression of phnO in plants would be useful in enhancing the phosphonate herbicide tolerance, particularly when AMPA is used as a herbicide or selective agent in plant transformation, and more particularly when glyphosate is used as a herbicide in combination with recombinant plants expressing a GOX gene.
Briefly therefore the present invention is directed to a composition of matter comprising a novel class of genes which encode proteins capable of N-acylation of phosphonate compounds and to methods of using these genes and encoded proteins for improving plant tolerance to phosphonate herbicides. The present invention is also directed to a method for selecting recombinant plants and microbes transformed with genes encoding proteins which are capable of N-acylation of phosphonate compounds, and to peptides which are capable of N-acylation of the compound N-amino-methyl-phosphonic acid (N-AMPA) and other related phosphonate compounds. In addition, the present invention is also directed to a method for using plants transformed with transacylase genes to prevent self-fertilization or to a method for enhancing hetero-fertilization in plants.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of producing stably transformed herbicide tolerant recombinant plants which have inserted into their genomes a polynucleotide sequence encoding a desired gene product, preferably an N-acyl-transferase enzyme. The polynucleotide sequence preferably is composed of a cassette containing a promoter sequence which is functional in plants and which is operably linked 5xe2x80x2 to a structural DNA sequence which, when transcribed into an RNA sequence, encodes an N-acyl-transferase enzyme peptide. The promoter sequence can be heterologous with respect to the structural DNA sequence and causes sufficient expression of the transferase enzyme in plant tissue to provide herbicide tolerance to the plant transformed with the polynucleotide sequence. The structural sequence is preferably operably linked 3xe2x80x2 to a 3xe2x80x2 non-translated polyadenylation sequence which functions in plants, and which when transcribed into RNA along with the structural sequence causes the addition of a polyadenylated nucleotide sequence to the 3xe2x80x2 end of the transcribed RNA. Expression of the structural DNA sequence produces sufficient levels of the acyltransferase enzyme in the plant tissue to enhance the herbicide tolerance of the transformed plant.
As a further embodiment, the structural DNA sequence may also contain an additional 5xe2x80x2 sequence encoding an amino-terminal peptide sequence which functions in plants to target the peptide produced from translation of the structural sequence to an intracellular organelle. This additional coding sequence is preferably linked in-frame to the structural sequence encoding the acyltransferase enzyme. The amino terminal peptide sequence can be either a signal peptide or a transit peptide. The intracellular organelle can be a chloroplast, a mitochondrion, a vacuole, endoplasmic reticulum, or other such structure. The structural DNA sequence may also be linked to 5xe2x80x2 sequences such as untranslated leader sequences (UTL""s), intron sequences, or combinations of these sequences and the like which may serve to enhance expression of the desired gene product. Intron sequences may also be introduced within the structural DNA sequence encoding the acyltransferase enzyme. Alternatively, chloroplast or plastid transformation can result in localization of an acyltransferase coding sequence and enzyme to the chloroplast or plastid, obviating the requirement for nuclear genome transformation, expression from the nuclear genome, and subsequent targeting of the gene product to a subcellular organelle.
Preferably, the recombinant plant expresses a gene encoding an enzyme which catalyzes the formation of AMPA. AMPA formation can result from the metabolism of a naturally occurring precursor, from a precursor such as glyphosate provided to the plant, or can result from the formation of AMPA through some catabolic pathway. Co-expression of GOX along with AMPA acyltransferase expression provides a plant which is surprisingly more resistant to certain phosphonate herbicides. However, one embodiment allowing plants transformed with only an N-acyltransferase to grow in the presence of AMPA or similar or related compounds would provide a useful selective method for identifying genetically transformed plants, callus, or embryogenic tissues.
In accordance with another aspect of the present invention is the provision of a method for selectively enhancing or improving herbicide tolerance in a recombinant plant which has inserted into its nuclear, chloroplast, plastid or mitochondrial genome a cassette comprised of a polynucleotide sequence which encodes an N-acyl-transferase enzyme.
A further embodiment encompasses the improvement of a method for selectively enhancing herbicide tolerance in a transformed plant expressing a GOX gene which encodes a glyphosate oxidoreductase enzyme expressed in the same plants in which an acyltransferase enzyme is produced.
In accordance with another aspect of the present invention is the provision of a method for producing a genetically transformed herbicide tolerant plant by inserting into a genome of a plant cell a cassette comprising a polynucleotide sequence which encodes an N-acyl-transferase enzyme.
A further embodiment encompasses the improvement of a method for producing a genetically transformed herbicide tolerant plant from a plant cell expressing a GOX gene which encodes a glyphosate oxidoreductase enzyme expressed in the same plant cell in which an acyltransferase enzyme is produced.
In any of the foregoing embodiments, the herbicide tolerant plant or plant cell can be selected from the group consisting of corn, wheat, cotton, rice, soybean, sugarbeet, canola, flax, barley, oilseed rape, sunflower, potato, tobacco, tomato, alfalfa, lettuce, apple, poplar, pine, eucalyptus, acacia, poplar, sweetgum, radiata pine, loblolly pine, spruce, teak, alfalfa, clovers and other forage crops, turf grasses, oilpalm, sugarcane, banana, coffee, tea, cacao, apples, walnuts, almonds, grapes, peanuts, pulses, petunia, marigolds, vinca, begonias, geraniums, pansy, impatiens, oats, sorghum, and millet.
In accordance with another aspect of the present invention is the provision of a peptide capable of N-acylation of the compound N-aminomethylphosphonic acid (N-AMPA or AMPA) or other such compounds which are capable of causing phytotoxic effects when applied to, introduced into, or produced by plant metabolisms. One such peptide is N-aminomethylphosphonic acid transacylase (AAT) derived from expression of an E. coli phnO structural gene sequence. Other peptides similar in structure and function to the E. coli phnO gene product are also contemplated.
Another aspect of the present invention is the provision of a method for selecting cells transformed with a vector containing an acyltransferase gene expressing an enzyme capable of N-acylation of AMPA and like compounds. The method includes the steps of transforming a population of cells with the vector, and isolating and purifying the transformed cells from non-transformed cells in the population after selecting for the transformed cells by incubation in the presence of amounts of AMPA sufficient to be inhibitory to the growth or viability of any non-transformed cells. The transformed cells can be bacterial, plant or fungal cells. Bacterial cells can be members of any of the families encompassed by Enterobacteraceae, Mycobacteraceae, Agrobacteraceae, and Actinobacteraceae, among others. Fungal cells can be members of Ascomycota, Basidiomycota, etc. Plant cells can be derived from any member of the Plantae family.
A further embodiment of the present invention provides for a method for producing a plant from a tissue, a cell, or other part of a plant which was derived from a plant transformed with an acyltransferase gene, a phnO gene, a gox gene, a gene in which GOX and acyltransferase peptides are produced from a translational fusion or a transcriptional fusion, or a polycistronic gene which encodes GOX and acyltransferase peptides.
A further embodiment of the present invention provides for a method for producing plants which express all or a portion of a phnO gene or similar acyltransferase gene, or a GOX gene as an antisense gene in a tissue specific manner.
Other aspects also include reagents such as antibodies directed to AMPA acyltransferase, and polynucleotides for use in identifying acyltransferase gene sequences. These reagents can be included in kits containing AMPA acyltransferase, polynucleotides which are or are complimentary to an AMPA acyltransferase gene sequence, polynucleotides for use in thermal amplification of an AMPA acyltransferase gene sequence, antibodies directed to AMPA acyltransferase for the detection of AMPA acyltransferase in the laboratory or in the field, and any other reagents necessary for use in kit form as well as for use in other assays contemplated herein.
A further object of the present invention is to provide a method for using phosphonate herbicides as chemical hybridizing agents. The method allows for selective gametocidal effects and for the production of male sterile plants. Such plants may be engineered so that gox or phnO, or gox and phnO fail to be expressed in plant tissues required for reproduction, causing sensitivity to applied phytotoxic compounds which inhibit formation of mature gamete structures.